Project Summary Asthma affects more than 300 million individuals worldwide, and more than half of patients with asthma have inadequately controlled symptoms and frequent exacerbations. Advances in our understanding of the immunology of asthma have led to the development of targeted biologic therapies against a single cytokine or receptor. Although these targeted therapies reduce exacerbations, they have not demonstrated disease modifying effects. Most asthma is allergic in origin, and the strongest risk factor for allergic asthma is allergy. Not all allergic patients have asthma, but many develop the disease over time, suggesting there are incremental and potentially reversible stages in the development of allergic asthma. Thus, identification of differences between allergic asthmatics (AA) and allergic non-asthmatic controls (AC) may provide insight into the mechanisms that ultimately lead to the development of asthma. The long-term goal of this research is to determine how dendritic cells (DC) orchestrate secondary immune responses in the lung and ultimately identify novel therapeutic targets aimed at inducing asthma remission. DC are critical regulators of the effector T cell response in the lung mucosa, and our previously published data suggest there are key differences in re-activation of allergen-specific T helper type 2 (Th2) cells during secondary responses in the lung. Therefore, we hypothesize that differences in airway DC between AA and AC determine the asthma phenotype. For this proposal, we will utilize bronchoscopic segmental allergen challenge (SAC) to mimic an asthma exacerbation in AA and AC and compare the immune response in the airway. Our preliminary data suggests that a subset of conventional DC in the bronchoalveolar lavage (BAL) may be more activated at baseline in AA compared to AC and that AA have higher levels of Th2- type cytokines and IgE in the airways following SAC. Furthermore, DC in AA may accumulate in the airway mucosa after allergen challenge. Finally, we demonstrate that the airway mucosa may be a separate immune microenvironment and identify the cellular components of inducible bronchus associated lymphoid tissue (iBALT) in mucosal samples. Based on these data we propose: Aim #1 to compare the phenotype of airway mucosal DC and BAL DC in AA and AC and Aim #2 to determine the transcriptional identity of airway mucosal immune cells in AA and AC. In Aim #1, we will compare the phenotype of DC isolated from BAL and the airway mucosa (using endobronchial brushing) of AA and AC at baseline and after SAC. We will also assess for known mediators of iBALT formation. In Aim #2, we will perform a comprehensive, unbiased transcriptional analysis of the immune cells in the airway mucosa using single-cell RNA sequencing (scRNAseq). We will also obtain endobronchial biopsies to assess for the presence of organized iBALT and validate the findings from scRNAseq. We believe these studies will advance our understanding of asthma pathogenesis by defining the relevant human lung DC subsets and providing insight into the airway microenvironment. Our results will form the basis of more targeted investigations using animal models, in vitro experiments and further translational human studies.